Production and characterization of intergeneric diploid cybrids derived from symmetric fusion between Microcitrus papuana Swingle and sour orange (Citrus aurantium)

Plants were regenerated from intergeneric somatic hybridization between embryogenic protoplasts of Microcitrus papuana Swingle and leaf-derived protoplasts of sour orange (Citrus aurantium L.) via electrofusion. The regenerated plants were morphologically similar to the leaf parent in growth vigor, leaf and branch structure. FCM analysis showed that they were diploids. Simple-sequence-repeat (SSR) and cleaved-amplified-polymorphic-sequence(CAPS) were employed for hybridity characterization. SSR banding patterns of the regenerated plants were identical to the leaf parent, sour orange, indicating that they possessed nuclear component derived from sour orange. DNA amplification with chloroplast and mitochondrial universal primers, followed by restriction endonuclease digestion, revealed polymorphism between the fusion parents. Therefore, this method was used to determine the cytoplasmic compositions of the regenerated plants. Banding patterns for all the polymorphic primer/enzyme combinations of the regenerated plants were similar to those of the embryogenic parent, M. papuana, suggesting that only the cytoplasmic components derived from the embryogenic parent were present in the regenerated plants. FCM, SSR and CAPS demonstrated that intergeneric diploid cybrids have been successfully obtained by symmetric fusion. Related results concerning nuclear and cytoplasmic composition of previous diploid somatic hybrids and potential mechanism for regeneration of such kind of plants are discussed herein. This revised version was published online in July 2006 with corrections to the Cover Date.     

A comparison of the somaclonal variation level of Rosa hybrida L. cv Meirutral plants regenerated from callus or direct induction from different vegetative and embryonic tissues

Summary Flowering plants of Rosa hybrida L. cv Meirutral have been obtained either from direct regeneration of adventitious shoots on leaf and root fragments, or through organogenesis and somatic embryogenesis on calli derived from anther, ovule, petal, sepal, receptacle, leaf, stem internode, root and zygotic embryo tissues. The calli derived from floral parts exhibited rhizogenesis. In this case direct induction of adventitious shoots from selected roots had to be performed in order to generate plants. A histological study of the morphogenetic calli was carried out. The plants regenerated directly and those regenerated from calli of leaf, stem internode, root and zygotic embryo tissues, together with reference plants propagated by cuttings, were compared on a phenotypic basis by taking into account petal number, form and colour, and plant growth habit. From these observations, it can be concluded that directly regenerated plants are as stable as reference plants while plants regenerated from callus are unstable, especially those derived from zygotic embryo tissues.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA3 gibberellic acid - IAA 3-indole-acetic acid - MS Murashige and Skoog - NAA 1-naphthalene acetic acid     

Somatic hybrids between Gossypium hirsutum L. (4×) and G. davidsonii Kellog (2×) produced by protoplast fusion

Somatic hybrids were obtained from electrofused protoplasts derived from embryogenic suspension cultures of tetraploid cotton (G. hirsutum L. cv. Coker 201) and embryogenic callus of diploid wild cotton G. davidsonii. The regenerants were initially identified as hybrids by RAPD (random amplified polymorphic DNA) analysis. Subsequently, observation on chromosome counting, morphology and SSR (simple sequence repeat) confirmed the hybrid status. Cytological investigation of the metaphase root-tip cells of the regenerated plants revealed there were 74 to 84 chromosomes in the plants, close to the expected 78 chromosomes. SSR analysis revealed the regenerated plants contained specific genomic fragments from both fusion partners, further confirmed their hybridity. The morphology of the plants was intermediate between the two fusion partners. The regenerants were difficult to develop into mature plants because their roots browned and they wilted from the stem apex before forming 3 to 5 true leaves. The hybrid plants were transferred to soil by grafting in vitro onto rootstocks.     

Ploidy Manipulation and Introgression of Resistance to Alternaria Helianthi from Wild Hexaploid Helianthus Species to Cultivated Sunflower (H. annuus L.) Aided by Anther Culture

The present investigation discusses the scope for transferring of resistance to leaf spot disease incited by Alternaria helianthi from two hexaploid wild species (H. tuberosus and H. resinosus) to diploid cultivated sunflower. Interspecific hybrids produced between sunflower and these two hexaploid species were partially fertile with tetraploid chromosome status. Backcrosses of these interspecific hybrids with cultivated sunflower resulted in the formation of sterile triploid plants. To overcome the problem of sterility and facilitate backcrosses with cultivated sunflower, anther culture of the tetraploid interspecific hybrids was carried out to bring down their chromosome number to diploid status. Anthers from both interspecific hybrids were cultured on basal Murashige and Skoog media supplemented with varying concentrations of organics and the growth regulators benzyladenine and naphthaleneacetic acid. Anthers of interspecific hybrids involving H. resinosus responded well and regenerated through an embryogenic route at a frequency of 98.7%. But in interspecific hybrids with H. tuberosus, anthers formed callus and subsequently regenerated shoots through an organogenic pathway. DNA ploidy analysis of anther culture plants of interspecific hybrids derived from H. tuberosus crosses was carried out to identify plants with desired diploid status. In vitro screening of parents, interspecific hybrids and anther culture plantlets against A. helianthi showed resistance in 68.5% of the anther culture plants of interspecific hybrids from H. tuberosus and in 24.3% of the plants derived from interspecific hybrids involving H. resinosus.     

Analysis of protoplast-derived plants of Saintpaulia ionantha H. Wendl

Of 3272 plants regenerated from protoplasts of 10 Saintpaulia ionantha genotypes, 98.4% survived transfer to the greenhouse. The frequency of regenerants with chlorophyll deficiencies, i.e. variegated leaves or albinos, was low (1.5%). There was a higher number of polyploid, in most cases tetraploid plants, regenerated from protoplasts (16%) which were identified by their altered morphology. Measurements of stomatal length and counting the number of chloroplasts per guard cell also allowed a clear differentiation between diploid and polyploid plants. The classification was confirmed by DNA content determination using flow cytometry. Mechanisms leading to polyploidization included spontaneous protoplast fusion as well as chromosome doubling during callus growth and shoot regeneration. Two genotypes with instabilities in flower colour showed completely altered flower colours in plants regenerated from protoplasts as well as in plants regenerated on leaf explants in vitro.     

Fertility and growth in the field of Lolium perenne and Festuca rubra plants regenerated from suspension cultured cells and protoplasts

The effect of regeneration of Lolium perenne and Festuca rubra from embryogenic suspension cells and protoplasts on fertility and growth was evaluated. Embryogenic suspension cultures were either routinely subcultured or cryopreserved and re-established. Phenology, morphology and fertility of regenerated plants were studied for two growing seasons in a replicated field experiment. Most regenerated L. perenne and F. rubra plants showed a delay in inflorescence emergence, a reduced seed yield and differences in morphological traits when compared with seed-grown plants. For L. perenne, performance of plants regenerated from cryopreserved suspension cultures and protoplasts was similar to that of respective plants regenerated from routinely maintained suspension cultures. However, differences in performance were observed for respective regenerants in F. rubra. The phenotypic deviation observed was partly reflected in the randomly amplified polymorphic DNA (RAPD) analysis performed. However, regenerants of both species showing similar, or even superior performance to the seed-grown plants were also found. Embryogenic suspension cells and corresponding protoplasts of L. perenne and F. rubra have the potential for producing fertile, well-performing plants which can be integrated in breeding programs.     

Field performance of embryogenic cell suspension-derived banana plants (Musa AAA, cv. Grande naine)

Growth and yield characteristics of two different clones of banana plants (Musa AAA cv. Grande naine) originating from four months old embryogenic cell suspensions were studied. These characteristics were compared with those plants produced by the conventional in vitro budding multiplication method. Two types of variants were observed during the acclimatization phase among 500 embryogenic cell suspension derived plants. The first type related to banana plants with `variegated or deformed leaves' were also observed in in vitro budding derived plants. The second type concerned `fasciated-leafed' plants. During the field growth, these two variant types produced plants morphologically similar to the other plants. Thus, none of the cell suspension derived plants exhibited off-type traits in the field. A Fisher block model was used to compare the field performances of the two clones produced through the two in vitro propagation techniques. The analysis of variance showed that there were no significant differences between the plants produced by either micropropagation techniques for the plant height and circumference, the length of the reference leaf, the number of nodal clusters of the inflorescence and of fruits, the bunch weight, the period of time between planting and flowering, and between planting and harvesting. This study showed that banana plants with an agronomical behaviour similar to those produced by the conventional in vitro budding method could be regenerated from embryogenic cell suspension. This revised version was published online in July 2006 with corrections to the Cover Date.     

Production of Primary Triticale from Calluses of Immature Abnormal Hybrid Embryos between Triticum durum and Secale cereale

Plant regeneration was attempted in callus induced from the immature abnormal hybrid embryos between T. durum and S. cereale, using 4-Huorophenoxyacetic acid as a growth regulator. In particular, the relationship of numerical variation in chromosomes between the callus tissues and the regenerated plants was investigated. Cytological observation revealed that there was no distinctive numerical difference between the shoot-forming (SF) and the non-shoot-forming calluses and also between the SF calluses and the regenerated plants. The root-tips of regenerated plants consisted of cells having various chromosome numbers, including the expected 2 n = 3 ×= 21 (genomes, ABR) of which the frequency was 69.8 %. The regenerated plants showed partial fertility, notwithstanding that the hybrid plants were expected to be sterile. Since the frequency of abnormal embryos was about 90 % in this cross, the utilization of abnormal embryos was demonstrated by use of callus culture.     

Somatic embryogenesis and plant regeneration of Tripsacum dactyloides L.

Summary This article reports the culture and plant regeneration of Tripsacum dactyloides. Mature embryos of Tripsacum dactyloides dactyloides were used to obtain embryogenic callus cultures. Currently, 180 normal plants have been regenerated from these cultures. Callus was initiated on MS medium supplemented with dicamba (10 mol or 20 mol) and sucrose (3% or 6%), and plants were regenerated on hormone free MS medium containing 2% sucrose. No significant differences were found in callus initiation frequency or in embryogenic response of cultures on the four combinations of sucrose and dicamba tested. The embryogenic cultures have been maintained for 9 months (12 subcultures) and have retained regeneration capacity. Plants regenerated from tissue culture of maize-by-Tripsacum hybrids could be useful in maize improvement.     

Particle-Bombardment-Mediated Co-Transformation of Maize with a Lysine Rich Protein Gene (sb401) from potato

Summary Little work has been reported on genetic transformation with maize inbred lines, especially elite inbred lines used in breeding. In this work, 7 self-pollinated inbred lines and 4 hybrid lines have been screened. The results revealed that calli derived from immature embryos from two inbred lines X333 and X301 were compact, hyperhydric and unsuitable for transformation, but the calli induced from other inbred lines and all the hybrid lines were friable and yellow and could be used for genetic transformation. The sb401 gene isolated from potato (Solanum berthaultii) encodes a protein with a high lysine content. Maize calli from 5 self-pollinated inbred lines and 4 hybrid lines were transformed using particle-bombardment with different plasmids to simultaneously introduce the sb401 lysine rich gene and the selectable gene hpt respectively. Two hundred and sixty-eight regenerated plants were obtained from these genotypes. Co-insertion was confirmed in 29 regenerated plants by PCR and Southern blot analysis. Transgene segregation of the R1 plants was observed and one marker-free transgenic maize line was recovered. Analysis of the crude protein content in mature seeds of R1 transgenic plants also showed an increase from 36.8% to 48.2%. This study thus provides a workable system for generating transgenic maize free from selectable marker genes and generates valuable resources for obtaining marker free transgenic maize with a high-lysine protein content.     

Analysis of genetic variability in various tissue culture-derived lemon plant populations using RAPD and flow cytometry

Five populations of lemon plants [Citrus limon (L.) Burm] obtained from undeveloped ovules through different tissue culture procedures were examined for the presence of somaclonal and irradiation-induced genetic variation. Tested groups were: (1) nucellar seedlings; (2) organogenic, regenerated via adventitious buds from nucellar seedling internodes; (3) embryogenic population, regenerated from non-irradiated nucellar callus via somatic embryogenesis; (4) embryogenic population, regenerated from irradiated nucellar callus via somatic embryogenesis; and (5) protoplast-derived, regenerated via somatic embryogenesis. Genomic DNA samples from 360 plants (72 from each group) were screened for polymorphism among RAPD fingerprints amplified by 10 decamer primers. Among all tested plants, genetic variation was detected only within the group of plants recovered from irradiated embryogenic calli. Out of 72 plants from that group, three had RAPD fingerprints different from the rest of the population, and fourth plant was found to be cytochimeric, consisting of diploid and tetraploid cells as revealed by flow cytometry. In all other populations of regenerated plants, we did not come across any plants with changed ploidy level.     

Plant Regeneration and Chromosomal Stability in Tissue Cultures of the Hybrids of Elymus canadensis with Psathyrostachys juncea and Secale cereale

Callus was induced from segments of immature inflorescences of Elymus canadensis×Psathyrostachys juncea and Elymus canadensis×Secale cereale F₁ hybrids on MS medium supplemented with 2 mg/l of 2,4-D and plants were regenerated from the calluses on a hormone free MS medium. The plants regenerated from both the hybrids exhibited a high degree of stability in morphology, chromosome number and chromosome pairing. The reasons for this are discussed.     

Selection in vitro for erucic-acid content in segregating populations of microspore-derived embryoids of Brassica napus

Microspore culture of Brassica napus under optimized conditions leads to the regeneration of microspore-derived embryoids that, at the late cotyledonary stage, contain large amounts of storage lipids, equal or similar in composition to those found in seeds of the homozygous donor plants. At that stage, the microspore-derived embryoids are large enough to allow the dissection of one cotyledon under aseptic conditions and the determination of its fatty-acid composition. The remaining part of the embryoid can be cultured further and regenerated to give a plant. This offers the possibility of early selection for fatty-acid composition in segregating populations of microspore-derived embryoids. In order to verify this hypothesis, embryoids were generated from microspores of F| plants derived from a cross between doubled haploid lines of the low-erucicacid cv. ‘Duplo’ and the high-erucic-acid cv. ‘Janetzki’. The contents of eicosenoic acid (C20: 1) and erucic acid (C22: 1) in the cotyledons and in the seeds derived from plants regenerated from the remaining parts of the embryoids were highly correlated (rs = 0.85**, P = 0.01). This indicates that, in breeding programmes for high erucic acid, the majority of the microspore—derived embryoids can be discarded at an early stage in vitro. Only microspore-derived embryoids with a high content of C20: 1+C22:1 in the cotyledons need to be transferred to the greenhouse. This report also deals with the addition of abscisic acid (ABA) to the embryoid culture medium to increase the correlation, and discusses the possible application of this system for the selection of high-oleic or low-linolenic types in corresponding microspore-derived embryoid populations.     

An assessment of morphogenic and transformation efficiency in a range of varieties of potato (Solanum tuberosum L.)

Summary To provide a truly genotype-independent transformation system, it is necessary to be able to transform a wide range of potato genotypes. The ability to regenerate shoots in vitro was determined for 34 potato varieties using tuber disc explants. Following a culture regime used extensively in previous studies with the variety Desiree, half of the varieties could be regenerated from tuber discs and half could not. From a sample of varieties that could be regenerated from tuber discs, all but one variety gave transgenic plants. Twelve varieties were evaluated for the capacity to regenerate shoots from leaf and internode explants excised from in vitro grown plants. All of the varieties tested regenerated adventitious shoots. Leaf and internode explants from 5 varieties were subsequently used for transformation, and transgenic plants were produced from two potato varieties that did not give transgenic plants from tuber disc explants. Some varieties could not be transformed by either method, and will require modification of the in vitro regeneration and transformation system to be successful.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid     

Heritable tissue culture induced variation in Zinnia marylandica

Summary Adventitious shoots of Zinnia marylandica, an amphidiploid with limited genetic segregation, were regenerated from cotyledonary tissue on Murashige-Skoog (MS) media containing 0.2 or 22.2 M thidiazuron (TDZ) and grown through flowering. Fisher's Test for Equal Variance indicated tissue culture induced plants had more variation than seed-derived control plants. Twelve of 149 (8%) plants derived from 0.2 M TDZ and three of 23 (13%) plants from 22.2 M TDZ had variant characters. Aberrant characteristics in self-pollinated variants included plant height, fertility, flower color and morphology, and were sexually transmitted, indicating genetic change had occurred. Aberrant characteristics not observed in regenerated plants arose in progeny.Abbreviation TDZ thidiazuron     

Variation amongst protoplast-derived moth bean Vigna aconitifolia plants

Summary Protoplasts were isolated from leaves of a single plant of Vigna aconitifolia, a drought resistant grain legume. The protoplasts regenerated and formed colony and calli from which 50 entire plants were regenerated and transferred to field conditions. Only 7 plants survived upto maturity and they flowered and produced pods with seed. The protoplast derived plants showed variation in important characters. Two groups of characters (one with 7 sets and another with 6 sets) were studied in the protoclones. In first group protoclones showed variations in seed germination, maturity age, pod length, pod and seed colour, number of abortive seed per pod and response to field rots, however, not much difference was recorded in pollen stainability and meiotic behaviour in these protoclones. In second group analysis of variants showed significance difference for plant height, rachis-length, length and breadth of mature odd leaflets, seed per pod and weight of seed. The results indicate that protoplast can be source of variation in this crop. However, detailed biochemical and genetical analysis of protoclones are required.     

大麦幼胚培养再生植株及其后代的细胞遗传学研究

从5个普通大麦品种的幼胚培养中均得到体细胞再分化植株。对214棵再生植株进行了细胞学检查,发现四倍体5株,占总数的2.3%;相互易位杂合体1株,占总数的0.46%。四倍体植株虽然得到少数大而瘪的种子,但它们次年没有发芽;易位杂合体减数分裂时的染色体构型为5Ⅱ+1Ⅳ,属于两对非同源染色体间的简单易位。其余所有二倍体植株的细胞     

Linamarin content and genetic stability of cassava plants derived by somatic embryogenesis

A protocol was established for high frequency cyclic somatic embryogenesis for different varieties of cassava. An efficient plant regeneration system was developed for the high cyanogenic variety PRC60a. Linamarin content and linamarase activity were determined in various tissues of secondary somatic embryos and regenerated plants of PRC 60a. Both linamarin and linamarase activity were not detected in embryogenic callus, roots induced from callus and somatic embryo tissues. The stems and leaves of regenerated plants (in vitro) and storage roots and leaves of mature plants (in vivo), however, contained variable amounts of linamarin and linamarase activity whereas in the non storage root tissues (in vitro) only linamarin was detected. The present study suggested that the linamarin biosynthetic pathway may be absent or not switched on in the embryogenic callus and somatic embryos. The ploidy level and somatic chromosome number of the regenerated plants were found to be same as the source plants. The availability of this regeneration system would be useful not only for investigating cyanogenesis but also for genetic manipulation in cassava. This revised version was published online in July 2006 with corrections to the Cover Date.     

Grafting in vitro Regenerated Pea (Pisum sativum L.) Shoots as a Method for Obtaining Mature Plants

A method of grafting pea plants is proposed to allow rapid availability of a large number of mature plants from in vitro regenerated shoots. Plant regeneration was obtained from macerated vegetative apices and immature embryos, but regenerated shoots rooted with low frequency (ca. 10 %). When those shoots were grafted onto seedling stocks of the same cultivar, 80 to 85 % of the grafts survived, grew to maturity and produced seed.     

Comparative Morphological Studies of Microspore Derived and F2 Plants Obtained from an Interspecific Rice Hybrid Oryza sativa Linn. ×O. rufipogon Griff.)*

Anther culture of an interspecific rice hybrid from a cross of Oryza sativa× O. rufipogon was attempted. Of the 117 regenerated pollen clones, 56 could survive to maturity. A majority of these were either haploids or doubled haploids and very few turned out to be chromosomal variants. Comparative study of doubled haploids and the seed derived F₂ plants indicate the distinct advantages of anther culture techniques. (1) Androgenic plants, though few in number, showed greater ariation for all the traits with the exception of ear bearing tillers. (2) Predominance of recombinants with wild traits was observed in F₂ segregation. (3) It was possible to recover indica type recombinants among the anther-derived plants with one or two traits introgressed from O. rufipogon. These results suggest the feasibility and utility of anther culture in distant hybridization for incorporation of alien variation into cultivated rice.